Neurotoxic Mechanisms of Reactive Nitrogen Intermediates

Children'S Hosp Of Philadelphia, Philadelphia PA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The detection of oxidized proteins, lipids and DNA/RNA in human as well as animal and cellular models of neurodegenerative diseases has been documented. In the last decade additional chemistry based on the formation of reactive nitrogen oxides generated by the versatile molecule nitric oxide have been also documented by the detection of tyrosine nitrated proteins mostly in the protein inclusions that characterize Parkinson's and other related diseases. Recent data also indicate that free tyrosine can be also converted to 3-nitrotyrosine or this unusual amino acid can be formed in the CNS following the degradation of nitrated proteins. Tyrosine is an important amino acid in the CNS since is the building block for the formation of dopamine. We hypothesized that the formation of 3-nitrotyrosine interferes with tyrosine metabolism and dopamine formation. Moreover given the structural similarities between 3-nitrotyrosine and dopamine we also hypothesized that this modified amino acid may also interfere with dopamine vesicular association as well as dopamine receptor signaling and re-uptake. We envision that 3-nitrotyrosine contributes in the de-regulation of dopamine in a manner that allows sufficient increase in the steady state of the free- non-vesicular dopamine, which allows for secondary formation of reactive species. Another unrecognized feature of 3-nitrotyrosine could be interference with mitochondrial functions leading to decline in ATP and production of reactive species. Preliminary data have also indicated a specific 3- nitrotyrosine dependent disruption of microtubule assembly leading in the formation of soluble and at later times insoluble aggregates. The presence of dopamine that could auto-oxidizes promoting formation of strong electrophiles and reactive species coupled with the decline in ATP and the formation of aggregates could promote cellular dysfunction and ultimately cell death. To test the critical aspects of these hypotheses we propose to evaluate the following: 1) determine if 3-nitrotyrosine interferes with the production and metabolism of dopamine, 2) evaluate if 3-nitrotyroinse interferes with mitochondrial respiration and mitochondrial function and 3) examine if the formation of a/[unreadable]-tubulin aggregates resulting from the specific incorporation of 3-nitroyrosine into cz-tubulin serve as a building block for the aggregation and/or fibrilization of a-synuclein and other proteins. An array of biochemical, pharmacological, molecular and electrophysiological approaches that are in place will be employed to performed the proposed experiments. The proposed experiments will evaluate the critical role of the endogenously generated unusual amino acid, 3-nitrotyrosine, as a central mediator of cellular dysfunction that leads to neurotoxicity and neurodegeneration. The application proposes to examine the alterations of fundamental regulatory pathways in dopamine metabolism, oxidative phosphorylation, and on protein aggregation, which constitute well-recognized molecular targets responsible for neuronal injury and death in Parkinson's disease. Insights into mechanisms of 3-nitrotyrosine toxicity will provide a novel and previously unrecognized molecular mechanism for the initiation and propagation of neuronal toxicity that culminates into a pathogenic phenotype and development of the disease. [unreadable] [unreadable]